1. Field of the Invention
The invention relates to covalent modification of proteins through their conjugation with other proteins. More particularly, the invention relates to the modulation of such conjugation involving the protein NEDD8.
2. Summary of the Related Art
Covalent modification of proteins through their conjugation with other proteins is an important biological mechanism for regulating protein metabolism and biological activity. Hershko and Ciechanover, Annu. Rev. Biochem. 61: 761-807 (1992) discloses conjugation of ubiquitin, one of the most conserved eukaryotic proteins, to other proteins through an enzymatic mechanism, as well as its role in protein degradation. Rock et al., Cell 78: 761-771 (1994) discloses that ubiquitination of protein antigens is required for processing of such antigens. Murray, Cell 81: 149-152 (1995), teaches that ubiquitination of cyclin is involved in cell cycle regulation. Scheffner et al., Cell 75: 495-505 (1993) discloses that ubiquitination of p53 is involved in degradation of this tumor suppressor.
The enzymatic pathway for ubiquitination has been reasonably well defined jentsch, Annu. Rev. Genet. 26: 179-207 (1992) discloses that ubiquitination requires initial activation of a conserved C-terminal glycine residue by the ubiquitin activating enzyme, E1, through formation of ubiquitin adenylate in an ATP-dependent process which liberates PPi, followed by thiol ester formation at a thiol site in E1 with release of AMP. Ubiquitin is then transferred to a thiol site in ubiquitin conjugating enzyme, E2, through formation of a thiol ester bond. Ubiquitin is then transferred to an epsilon amino group of a lysine residue in the target protein through an amide linkage, usually with the involvement ofubiquitin-protein isopeptide ligase, E3. Hopkin J. Natl. Inst. Health Res. 9: 36-42 (1997), teaches that target specificity is regulated by the particular combination of E2 and E3 protein with more than 30 E2 proteins and 10 E3 proteins being known at present.
Ubiquitin is not the only protein which is used to modify other proteins through covalent linkage, however. Kamitani et al., J. Biol. Chem. 272: 14001-14004 (1997), discloses the sentrin, a ubiquitin-like protein, appears to be processed similarly to ubiquitin, but has a smaller target protein repertoire than ubiquitin. Okura et al., J. Immunol. 272: 4277-4281 (1986) teaches that sentrin protects cells against anti-FAS and tumor necrosis factor-mediated cell death. Loeb and Haas, J. Biol. Chem. 267: 7806-7813 (1982), discloses that ubiquitin cross-reactive protein (UCRP), which contains two ubiquitin domains, is conjugated to a large number of intracellular proteins. Kumar et al., Biochem. Biophys. Res. Commun. 185: 1155-1161 (1992), discloses another ubiquitin-like protein, called NEDD8, for Neural precursor cell-Expressed Developmentally Down regulated. Kamitani et al., J. Biol. Chem. 272: 28557-28562 (1997), teaches that NEDD8 is predominantly expressed in the nucleus and is conjugated to target proteins through a mechanism analogous to ubiquitination.
These proteins, which covalently modify other cellular proteins, are important components of biological regulatory processes. The nuclear expression pattern and developmental regulation of NEDD8 make it a particularly compelling candidate as an important regulatory molecule. There is a need, therefore to understand the role of NEDD8 in biological regulation. Unfortunately, the lack of understanding about the specific proteins involved in NEDD8 conjugation has resulted in a lack of effective tools to probe the role of NEDD8. There is, therefore, a need for better tools to utilize in elucidating the role of NEDD8 in biological regulation. Ideally, such tools would allow modulation of the activation and/or conjugation of NEDD8.
The invention provides compositions and methods for detecting and/or modulating the conjugation of NEDD8 and/or its transfer to a target protein, as well as compositions and methods for discovering molecules which are useful in detecting and/or modulating the conjugation of NEDD8 and/or its transfer to a target protein. The present invention arises from the purification and characterization of novel NEDD8 activating and conjugating enzymes.
In a first aspect, the invention provides purified NEDD8-activating protein beta subunit (NAE1-beta). The primary amino acid sequence of a preferred embodiment of such NAE1-beta protein is shown in FIG. 1.
A second aspect, the invention provides NAE1-beta expression elements Such elements include, without limitation, isolated of recombinant nucleic acid sequences encoding NAE1-beta or nucleic acid sequences specifically homologous or specifically complementary thereto, vectors comprising any such nucleic acid sequences and recombinant expression units which express NAE1-beta, or, antisense transcripts or dominant negative mutants thereof.
The purified protein and its structural information provided herein enables the preparation of NAE1-beta-binding molecules (NAE1BBMs). Thus, in a third aspect, the invention provides methods for identifying NAE1BBMs. One preferred method according to this aspect of the invention comprises screening for NAE1BBMs by contacting purified NAE1-beta according to the invention and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to NAE1-beta. Another preferred method according to this aspect of the invention comprises rationally designing molecules to bind NAE1-beta based upon structural information from the purified NAE1-beta protein provided by the invention and determining whether such rationally designed molecules bind specifically to NAE1-beta. This aspect of the invention includes NAE1BBMs identified by the methods according to the invention.
NAE1BBMs can be used in conventional assays to detect the presence or absence, and/or quantity of NAE1-beta, NAE1 heterodimer, or NAE1 heterodimer/NEDD8 complex in a biological sample. Thus, in a fourth aspect, the invention provides methods for determining the presence or absence and/or quantity of NAE1-beta, NAE1 heterodimer, or NAE1 heterodimer/NEDD8 complex in a biological sample. Such methods comprise providing a detectable NAE1BBM to a biological sample, allowing the detectable NAE1BBM to bind to NAE1-beta, NAE1 heterodimer, or NAE1 heterodimer/NEDD8 complex, if any is present in the biological sample, and detecting the presence or absence and/or quantity of a complex of the detectable NAE1BBM and NAE1-beta, NAE1-heterodimer, or NAE1 heterodimer/NEDD8 complex.
Nucleic acid sequences specifically complementary to and/or specifically homologous to nucleic acid sequences encoding NAE1-beta can also be used in conventional assays to detect the presence or absence of NAE1-beta nucleic acid in a biological sample. Thus, in a fifth aspect, the invention provides methods for determining the presence or absence and/or quantity of NAE1-beta nucleic acid in a biological sample. In preferred embodiments, such assays are nucleic acid hybridization and/or amplification assay, such assays comprising providing to the biological sample a nucleic acid sequence which is specifically complementary to NAE1-beta nucleic acid.
In a sixth aspect, the invention provides methods for identifying modulating ligands of NAE1-beta. Some NAE1BBMs are capable of acting as antagonists or agonists of NAE1-beta. Thus, the method according to this aspect of the invention comprises providing NAE1BBMs to an assay system for NAE1-beta participation in the NEDD8-activation/conjugation pathway, and determining whether such NAE1BBMs interfere with or enhance the ability of NAE1-beta to participate in the NEDD8-activation/conjugation pathway. The NAE1BBMs are preferably provided as a population of molecules (most preferably rationally designed molecules), or as a mixed population of molecules, as for example in a screening procedure. This aspect of the invention includes modulating ligands of NAE1-beta identified by this method according to the invention.
In a seventh aspect, the invention provides modulating ligands of NAE1-beta. Preferred modulating ligands are NAE1BBMs which act as antagonists, interfering with the ability of NAE1-beta to participate in the NEDD8-activation/conjugation pathway. Other preferred modulating ligands are NAE1BBMs which act as agonists, enhancing the ability of NAE1-beta to participate in the NEDD8-activation/conjugation pathway. In certain embodiments, such NAE1BBMs preferably interact with NAE1-beta to inhibit or enhance the formation of NAE1 heterodimer, the formation of NEDD8 adenylate, the formation of a thiol ester bond between NEDD8 and NAE1, and/or transfer of NEDD8 to NEDD8-conjugating enzyme.
In an eighth aspect, the invention provides methods for modulating the activation and/or conjugation of NEDD8. One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand of NAE1-beta or a recombinant expression unit which expresses NAE1-beta or an antagonist thereof to a biological system in which NEDD8 is conjugated to another protein.
In a ninth aspect, the invention provides oligonucleotides that are specifically complementary to a portion of a nucleotide sequence shown in FIG. 1. Preferred embodiments include hybridization probes and antisense oligonucleotides.
In a tenth aspect, the invention provides methods for identifying NAE1-alpha binding molecules (NAE1ABMs). The present inventors have identified the alpha subunit of the NAE1 heterodimer (NAE1-alpha). Surprisingly, it has an amino acid sequence which is substantially identical to a protein previously identified as amyloid precursor protein binding protein 1 (APP-BP1; see Chow et al., J. Biol. Chem. 271: 11339-11346 (1996)). One preferred method according to this aspect of the invention comprises screening for NAE1ABMs by contacting purified NAE1-alpha and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to NAE1-alpha. Another preferred method according to this aspect of the invention comprises rationally designing molecules to bind NAE1-alpha based upon structural information from the NAE1-alpha protein identified by the present inventors and determining whether such rationally designed molecules bind specifically to NAE1-alpha. This aspect of the invention includes NAE1ABMs identified by the methods according to the invention.
NAE1ABMs cn be used in conventional assays to detect the presence or absence, and/or quantity of NAE1-alpha, NAE1 heterodimer, or NAE1 heterodimer/NEDD8 complex in a biological sample. Thus, in an eleventh aspect, the invention provides methods for determining the presence of absence and/or quantity of NAE1-alpha, NAE1 heterodimer, or NAE1 heterodimer/NEDD8 complex in a biological sample. Such methods comprise providing a detectable NAE1ABM to a biological sample, allowing the detectable NAE1ABM to bind to NAE1-alpha, NAE1 heterodimer, or NAE1 heterodimer/NEDD8 complex, if any is present in the biological sample, and detecting the presence or absence and/or quantity of a complex of the detectable NAE1ABM and NAE1-alpha, NAE1-heterodimer, or NAE1 heterodimer/NEDD8 complex. In preferred embodiments, the method according to this aspect of the invention is used to detect the presence or absence, and/or quantity of NAE1 heterodimer or NAE1 heterodimer/NEDD8 complex in a biological sample.
Nucleic acid sequences specifically complementary to and/or specifically homologous to nucleic acid sequences encoding NAE1-alpha can also be used in conventional assays to detect the presence or absence of NAE1-alpha nucleic acid in a biological sample in which NEDD8 conjugation is suspected. Thus, in a twelfth aspect, the invention provides methods for determining the presence or absence and/or quantity of NAE1-alpha nucleic acid in such a biological sample. In preferred embodiments, such assays are nucleic acid hybridization and/or amplification assays, such assays comprising providing to the biological sample a nucleic acid sequence which is specifically complementary to NAE1-alpha nucleic acid.
In an thirteenth aspect, the invention provides methods for identifying modulating ligands of NAE1-alpha. Some NAE1ABMs are capable of acting as antagonists or agonists of NAE1-alpha. Thus, the method according to this aspect of the invention comprises providing NAE1ABMs to an assay system for NAE1-alpha participation in the NEDD8-activation/conjugation pathway, and determining whether such NAE1ABMs interfere with or enhance the ability of NAE1-alpha to participate in the NEDD8-activation/conjugation pathway. The NAE1ABMs are preferably provided as a population of molecules (most preferably rationally designed molecules), or as a mixed population of molecules, as for example in a screening procedure. This aspect of the invention includes antagonists or agonists of NAE1-alpha identified by this method according to the invention.
In a fourteenth aspect the invention provides a purified complex of NAE1-beta and NAE1-alpha, or of NAE1-beta, NAE1-alpha and NEDD8, or a purified complex of portions thereof.
In a fifteenth aspect, the invention provides modulating ligands of NAE1-alpha. Certain preferred modulating ligands are NAE1ABMs which act as antagonists which interfere with the ability of NAE1-alpha to participate in the NEDD8-activation/conjugation pathway. Other preferred modulating ligands are NAE1ABMs which act as agonists which enhance the ability of NAE1-alpha to participate in the NEDD8-activation/conjugation pathway. Preferably, such inhibition or enhancement is specific, as described above. In certain embodiments, such modulating ligands preferably interact with NAE1-alpha to inhibit or enhance the formation of NAE1 heterodimer, the formation of NEDD8 adenylate, the formation of a thiol ester bond between NEDD8 and NAE1, and/or transfer of NEDD8 to NEDD8-conjugating enzyme.
In a sixteenth aspect, the invention provides methods for modulating the activation and/or conjugation of NEDD8. One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand NAE1-alpha or a recombinant expression unit which expresses NAE1-alpha or an antagonist thereof to a biological system in which NEDD8 is conjugated to another protein.
In a seventeenth aspect, the invention provides alleic varients of NAE-1 alpha. This aspect of the invention further includes NAE1-alpha allelic variant expression elements. Such elements include, without limitation, isolated or recombinant nucleic acid sequences encoding NAE1-alpha, or nucleic acid sequences specifically homologous or specifically complementary thereto, vectors comprising any such nucleic acid sequences, and recombinant expression units which express NAE1-beta or antisense transcripts or dominant negative mutants thereof.
In a eighteenth aspect, the invention provides methods for modulating auxin response in plants. The present inventors have discovered that NAE1-alpha shares 39% identity and 61% conserved residues with Aux1 in A. Thaliana, which is involved in signal transduction in the auxin response in plants. This suggests that antagonists of NAE1-beta and/or NAE1-alpha should down-regulate the auxin response, and that expression of NAE1-beta and/or NAE1-alpha should up-regulate the auxin response. One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand of NAE1-beta or NAE1-alpha or a recombinant expression unit which expresses NAE1-beta or NAE1 or an antagonist thereof to a plant that is under auxin treatment.
In a nineteenth aspect, the invention provides methods for modulating the biological role of APP and/or beta peptide accumulation in a biological system. The present inventors have discovered that NAE1-alpha is substantially the same protein is amyloid precursor protein binding protein-1 (APP-BP1). This suggests that antagonists or agonists of NAE1-beta and/or NAE1-alpha should modulate APP function, including its role in beta peptide accumulation. One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand of NAE1-beta or NAE1-alpha or a recombinant expression unit which expresses NAE1-beta or NAE1 or an antagonist thereof to a biological system.
In an twentieth aspect, the invention provides two new purified NEDD8-conjugating enzymes and allelic variants thereof. The primary amino acid sequence of a preferred embodiment of a first such NEDD8-conjugating enzyme (NCE1) is shown in FIG. 2. The primary amino acid sequence of a preferred embodiment of a second such NEDD8-conjugating enzyme (NCE2) is shown in FIG. 5.
In a twenty-first aspect, the invention provides NEDD8-conjugation enzyme expression elements. Such elements include, without limitation, isolated or recombinant nucleic acid sequences encoding NCE1 or NCE2 or dominant negative mutants thereof, or capable of expressing antisense transcripts thereof or nucleic acid sequences specifically homologous or specifically complementary thereto, and vectors comprising any such recombinant expression elements, preferably expression vectors.
The purified protein and is structural information provided herein enables the preparation of NCE1 and NCE2 binding molecules, respectively NCE1BMs and NCE2BMs. Thus, in a twenty-second aspect, the invention provides methods for identifying NCE1BMs and NCE2BMs. One preferred method according to this aspect of the invention comprises screening for NCE1BMs or NCE2BMs by contacting purified NCE1 or NCE2 according to the invention and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to NCE1 or NCE2. Another preferred method according to this aspect of the invention comprises rationally designing molecules to bind NCE1 or NCE2 based upon structural information from the purified NCE1 and NCE2 provided by the invention and determining whether such rationally designed molecules bind specifically to NCE1 to NCE2. This aspect of the invention includes NCE1BMs and NCE2BMs identified by the method according to the invention.
NCE1BMs and NCE2BMs can be used in conventional assays to detect the presence or absence, and/or quantity of NCE1 or NCE2, or NCE1 or NCE2/NEDD8 complex in a biological sample. Thus, in a twenty-third aspect, the invention provides methods for determining the presence or absence and/or quantity of NCE1 or NCE2, or NCE1 or NCE2/NEDD8 complex in a biological sample. Such methods comprise providing a detectable NCE1BM or NCE2BM to a biological sample, allowing the detectable NCE1BM or NCE2BM to bind to, respectively NCE1 or NCE2, or respectively NCE1 or NCE2/NEDD8 complex, if any is present in biological sample, and detecting the presence or absence and/or quantity of a complex of the detectable NCE1BM or NCE2BM and NCE1 or NCE2, or NCE1 or NCE2/NEDD8 complex.
Nucleic acid sequences specifically complementary to and/or specifically homologous to nucleic acid sequences encoding NCE1to NCE2 can also be used in conventional assays to detect the presence or absence of NCE1 or NCE2 nucleic acid in a biological sample. Thus, in a twenty-fourth aspect, the invention provides methods for determining the presence or absence and/or quantity of NCE1 or NCE2 nucleic acid in a biological sample. In preferred embodiments, such assays are nucleic acid hybridization and/or amplification assays, such assays comprising providing to the biological sample a nucleic acid sequence which is specifically complementary to NCE1 or NCE2 nucleic acid.
In a twenty-fifth aspect, the invention provides methods for identifying modulating ligands of NCE1 or NCE2. Some NCE1BMs or NCE2BMs are capable of acting as antagonists or agonists of, respectively NCE1 or NCE2. Thus, the method according to this aspect of the invention comprises providing NCE1BMs or NCE2BMs to an assay system for NCE1 or NCE2 participation in the NEDD8-activation/conjugation pathway, and determining whether such NCE1BMs or NCE2BMs interfere with or enhance the ability of NCE1 or NCE2 to participate in the NEDD8-activation/conjugation pathway. The NCE1BMs or NCE2BMs are preferably provided as a population of molecules (most preferably rationally designed molecules), or as a mixed population of molecules, as for example in a screening procedure. This aspect of the invention includes modulating ligands of NCE1 or NCE2 identified by this invention according to the invention.
In a twenty-sixth aspect, the invention provides modulating ligands of NCE1or NCE2. Preferred modulating ligands are NCE1BMs or NCE2BMs which act as antagonists, interfering with the ability of NCE1 or NCE2 to participate in the NEDD8-activation/conjugation pathway. Other preferred modulating ligands are NCE1BMs or NCE2BMs which act as agonists, enhancing the ability of, respectively NCE1 or NCE2 to participate in the NEDD8-activation/conjugation pathway. In certain embodiments, such NCE1BMs or NCE2BMs preferably interact with NCE1 or NCE2 to inhibit or enhance the formation of a thiol ester bond between NEDD8 and NCE1 to NCE2 and/or transfer of NEDD8 to its target protein.
In a twenty-seventh aspect, the invention provides methods for modulating the conjugation of NEDD8 or its transfer to a target protein. One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand of NCE1 or NCE2 or a recombinant expression unit which expresses NCE1 or NCE2 or an antagonist thereof to a biological system in which NEDD8 is conjugated to another protein.
In a twenty-eighth aspect, the invention provides oligonucleotides that are specifically complementary to a portion of a nucleotide sequence shown in FIG. 2 or FIG. 5. Preferred embodiments include hybridization probes and antisense oligonucleotides.